Jacob, The solubility of the alpha anomer of DDM is actually not bad (it's only really bad for the alkyl glucosides), just the phase behavior is different. Still getting pure beta anomer is the tough problem, which is part of the reason it is so expensive.
Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** On Apr 27, 2012, at 10:54 AM, Jacob Keller wrote: > Wouldn't the lack of solubility of the alpha form of DDM suggest an easy > purification protocol for the beta form? > > JPK > > On Fri, Apr 27, 2012 at 8:40 AM, R. M. Garavito <rmgarav...@gmail.com> wrote: > Hongjun, > > I am in agreement with Bert as DDM is exceedingly difficult to crystallize, > even in organic solvents. This is one of the reasons it is so expensive. > However, you can produce a lot of "quasi"-crystals that do show low > resolution diffraction. As Bert said, you may have protein/detergent > crystals that are just poorly ordered. I would disagree with Bert only > slightly concerning these crystals in that while you might suppose that most > or all of the lattice contacts are mediated by detergent and not by protein. > Instead, you might also be observing protein-protein contacts being disturbed > by less than optimal detergent contacts (either detergent-detergent or > detergent-protein). Try changing the detergent to decyl-maltoside (DM) to > see if you get similar results. It was the change from DDM to DM that really > gave great crystals for the 13-subunit bovine cytochrome c oxidase. > > Another thing to watch out for is the dreaded contamination factor, either by > protein or detergent. I have seen cases where crystals were from a > contaminating protein (such as those which may bind to Ni-affinity resin and > are not separated by gel filtration) at as low as 1% (by weight) > contamination. More insidious is detergent contamination. DDM is is the > beta anomer, but all batches are contaminated with varying amounts of the > alpha anomer. The alpha anomers of alkyl glycoside detergents tend to > crystallize much more readily than the beta anomers. Despite their best > efforts, manufacturers occasionally produce batches with a high level of > alpha anomer contamination. I have personally tested a batch of beta-octyl > glucoside (from a very reputable company) that did not dissolve; other > batches from a different company were cloudy when making a 10% stock > solution. Alpha-octyl glucoside is not soluble below ~32C and make some very > nice crystals in aqueous solution at room temperature. So try a batch of DDM > from another source. > > Best of luck, > > Michael > > **************************************************************** > R. Michael Garavito, Ph.D. > Professor of Biochemistry & Molecular Biology > 603 Wilson Rd., Rm. 513 > Michigan State University > East Lansing, MI 48824-1319 > Office: (517) 355-9724 Lab: (517) 353-9125 > FAX: (517) 353-9334 Email: rmgarav...@gmail.com > **************************************************************** > > > > > On Apr 26, 2012, at 5:32 PM, Van Den Berg, Bert wrote: > >> Generally speaking it is quite hard to crystallize DDM since it is so >> soluble (>20% in water). You most likely have protein crystals (of course >> containing a lot of detergent as well) that are just not ordered, presumably >> because most or all of the lattice contacts are mediated by detergent and >> not by protein. Unfortunately this is the norm for membrane protein >> crystallization. >> >> Good luck, Bert >> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of 于洪军 >> [hongju...@moon.ibp.ac.cn] >> Sent: Friday, April 27, 2012 6:07 AM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: [ccp4bb] detergent crystal? >> >> >> Hi, >> >> I am trying to screen crystals of membrane protein in DDM solutions. I got >> crystals and its diffraction pattern as I enclosed. Membrane protein >> crystallization seems quite different from soluble protein. The condition >> contains PEG400. I learn from other topic here that PEG400 can easily >> produce DDM detergent crystals. Is it detergent crystal ? Can I tell this >> from the diffraction pattern? Advices would be greatly appreciated. Thank >> you. >> >> >> Hongjun > > > > > -- > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > email: j-kell...@northwestern.edu > *******************************************
